title: A new method of high-speed cellular protein separation and insight into subcellular compartmentalization of proteins.
abstract: Transglutaminase (TGM)-2 is a ubiquitous protein with important cellular functions such as regulation of cytoskeleton, cell adhesion, apoptosis, energy metabolism, and stress signaling. We identified several proteins that may interact with TGM-2 through a discovery-based proteomics method via pull down of flag-tagged TGM-2 peptide fragments. The distribution of these potential binding partners of TGM-2 was studied in subcellular fractions separated by density using novel high-speed centricollation technology. Centricollation is a compressed air-driven, low-temperature stepwise ultracentrifugation procedure where low extraction volumes can be processed in a relatively short time in non-denaturing separation conditions with high recovery yield. The fractions were characterized by immunoblots against known organelle markers. The changes in the concentrations of the binding partners were studied in cells expressing short hairpin RNA against TGM-2 (shTG). Desmin, mitochondrial intramembrane cleaving protease (PARL), protein tyrosine kinase (NTRK3), and serine protease (PRSS3) were found to be less concentrated in the 8.5%, 10%, 15%, and 20% sucrose fractions (SFs) from the lysate of shTG cells. The Golgi-associated protein (GOLGA2) was predominantly localized in 15% SF fraction, and in shTG, this shifted to predominantly in the 8.5% SF and showed larger aggregations in the cytosol of cells on immunofluorescent staining compared to control. Based on the relative concentrations of these proteins, we propose how trafficking of such proteins between cellular compartments can occur to regulate cell function. Centricollation is useful for elucidating biological function at the molecular level, especially when combined with traditional cell biology techniques.
authors.affiliation: Ocular Wound Healing and Therapeutics Laboratory, Singapore Eye Research Institute, Singapore.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Png
authors.forename: WanWen
authors.initials: W
authors.lastname: Lan
authors.forename: Melisa
authors.initials: M
authors.lastname: Lazaroo
authors.forename: Silin
authors.initials: S
authors.lastname: Chen
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
authors.forename: Louis
authors.initials: L
authors.lastname: Tong
mesh_terms: D000818:Animals; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013347:Subcellular Fractions / Q000378:metabolism*; D011503:Transglutaminases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism*; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D011506:Proteins; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins